Automated Solid Phase Extraction of Allantoin from Cosmetics and

Topical Pharmaceuticals Prior to Analysis by HPLC

Keywords: Allantoin, Automation, CHROMABOND HR-XA, Cosmetics, Glyoxylic Acid Diureide, GX-271 ASPEC,
HILIC, HPLC, Ointments, Pharmaceuticals, Sample Preparation, Solid Phase Extraction, SPE, Topical
Pharmaceuticals, 5-Ureidohydantoin
This study was performed by Dr. Martin Roedel and colleagues at Macherey-Nagel GmbH in Dueren, Germany.
Introduction
Allantoin is a heterocyclic organic compound derived from purine (Figure 1). It is also referred to as Glyoxylic
Acid Diureide or 5-Ureidhyantoin. Allantoin is a metabolic end product of purine degradation in mammals (with
the exception of humans and higher apes), as well as a metabolic intermediate in plants and some bacteria
(Young et al., 1944; Fujiwara et al., 1995). Allantoin can be found in many plant species.
Comfrey (Symphytum officinale) has particularly high levels of allantoin.

Figure 1. Chemical Structure of Allantoin (CAS no. 97-59-6).

Allantoin has a long history of use in a variety of topical pharmaceuticals and cosmetics for skin care. It has
keratolytic, moisturizing, soothing and anti-irritant properties. Allantoin promotes the renewal of epidermal cells
and accelerates wound healing (AKEMA, 2008). Allantoin is used in pharmaceuticals and dermatologic products
in the treatment of ulcers, slow-healing wounds, burns, psoriasis and dry skin. The U.S. Food and Drug
Administration (FDA) has classified allantoin as a Category 1 (Safe and Effective) active ingredient for skin
protection (Federal Register, 1983, 1990) at use levels of 0.5% to 2.0%.
Allantoin is used in a variety of cosmetic products such as skin creams, lip-care products, powders, suntan and
sunburn lotions, hair care products, diaper rash ointments and mouthwashes. Recommended levels in cosmetics
are from 0.1% to 0.5% (Thornfeldt, C., 2005).
There is a great deal of interest from pharmaceutical and cosmetic manufacturers in determining the amount of
allantoin in a variety of products. This application note describes an automated SPE method for the extraction of
allantoin from a cosmetic product. Allantoin was extracted from a cosmetic product using the automated Gilson
GX-271 ASPEC System (Figure 2). Details of this procedure are described below. Allantoin levels were
determined using HPLC and a NUCLEODUR 100-3 HILIC column.

Figure 2. Gilson GX-271 ASPEC System with 406 Single Syringe Pump (part no. 2614007).
Experimental Conditions
Materials
All solvents used were HPLC grade. All reagents were ACS grade or better. Allantoin (>98% purity) was obtained
from Fluka Analytical (part no. 05670). Macherey-Nagel CHROMABONDHR-XA cartridges, 60mg/3mL (Part
no. 730950) were used to extract the allantoin from the cosmetic product. The cosmetic products were obtained
from a cosmetic manufacturer in Europe. One product had an unknown amount of allantoin. The second product
was allantoin free. The allantoin free product was spiked with 5 mg of allantoin.
Preparation of Sample Prior to Solid Phase Extraction and Additional Liquid Handling Steps
One gram of allantoin sample was mixed with 100 mL of ultra-pure water.
Automated Solid Phase Extraction
The Gilson GX-271 ASPEC System was configured as follows:
Description

Part Numbers

GX-271 ASPEC w/ Single 406 Syringe Pump

2614007

10 mL Syringe
SPE Pressure Reg. Assembly and Plumbing package for gas +
10 mL Plumbing Package
221 x 1.5 x 1.1 BV Tapered Probe and
Guide Assembly for 1.5 mm Probes

25025345
25051376, 2644703, and 2644701
27067374 and 26046228

Rinse Stations
Locator Tray for Five 20-series Racks
DEC Accessory Set for 3 mL SPE Cartridges

26034551 and 26034555

26041033
2604702

Rack Code 343 for 80 13 x 100mm Tubes

260440025

Rack Code 345 for 44 16 x 150mm Tubes

Solvent Reservoir Tray Insert for 700 mL bottles and pkg of four
700 mL solvent bottles

260440041
260440005 and 543701700

Safety Shield Assembly, GX27X

TRILUTION LH Software Package

2604706
21063020, 210630R20 and ORACLE10GXE

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The SPE procedure used 60 mg/ 3mL CHROMABONDHR-XA solid phase extraction cartridges (Macherey-Nagel,
Germany). The cartridges were sealed using Gilson 3 mL Sealing Caps.
The solid phase extraction and liquid handling protocol is entirely automated using the Gilson GX-271 ASPEC
system.
The SPE steps are summarized with the general schematic provided in the GX-271 ASPEC control software,
TRILUTION LH (Figure 3).

Figure 3. TRILUTION LH Basic SPE Tasks for Solid Phase Extraction of Allantoin from a Cosmetic Product
The summary of each step are as follows:
Initialization Step: Gilson Mobile SPE Racks are moved above the waste rack (Figure 4)
Condition the cartridge with 1 mL of methanol at 0.5 mL/min
Condition the cartridge with 1 mL of ammonia, w(NH3) = 5% at 0.5 mL/min
Dispense 4 mL of sample( 1g in 100 mL water) into a tube at 5 mL/min
Dispense 400 L ammonia, w(NH3) = 26% at 0.5 mL/min into the same tube as step above
Load 1.1 mL of the sample mix created above onto the SPE cartridge at 0.5 mL/min
Wash cartridge with 1 mL of ammonia, w(NH3) = 5% at 0.5 mL/min
Wash cartridge with 1 mL of methanol at 0.5 mL/min
Dry with 5 mL air, 3 mL/min
Move the Gilson Mobile SPE Rack over the collection tubes
Elute with 2X 600uL Hydrochloric acid, HCl, 0.1 mol/L at 0.5 mL/min
Eluent can be injected directly into the HPLC system

Figure 4. Gilson Mobile SPE Rack

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HPLC Analysis
Allantoin concentrations in the extracts were analyzed using high-performance liquid chromatography
(Dionex P680, USA) with UV detection (Dionex UVD 17U, USA) using the following conditions:
Column:
Conditions:

Detection :

Macherey-Nagel EC 125/3 NUCLEODUR 100-3 HILIC (Part no. 760 531.30)

Eluent A:
10 mmol/L Ammonium chloride, pH 3.0
20
Eluent B:
Acetonitrile
80
Flow Rate:
0.3 mL/min
Temperature:
Ambient
Injection Volume:
20 L
Concentration:
(Allantoin) = 5 g/mL Eluent
UV, 214 nm

Results

Figure 5. Chromatogram of Allantoin from Cosmetic Product Following

SPE Purification. The Retention Time for Allantoin is 3.66 minutes.
The recovery of allantoin from the cosmetic product (n=3) was 85.5%
Conclusion
An effective automated SPE method was developed for the extraction of allantoin from cosmetic/pharmaceutical
products. The use of the Gilson GX-271 ASPEC in combination with CHROMABOND HR-XA SPE cartridges and a
NUCLEODUR 100-3 HILIC HPLC column resulted in excellent recovery rates.
Using the Gilson GX-271 ASPEC for automation of the solid phase extraction (SPE) process increased sample
throughput, reduced solvent usage and reduced the potential errors that may occur in during manual
processing of samples. Automation also permitted scientists to spend more time planning scientific experiments
and developing new methods for the analysis of compounds of interest to the laboratory.

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References
AKEMA srl (an allantoin manufacturer) website (2008). Allantoin_CFTA.doc. 27/02/2008 Version 1.
http://www.akema.it/allantoin.htm
Federal Register (1983, 1990). FDA Monograph on Skin Protectant Drug Products for Over-the Counter (OTC)
Human Use. Volume 48, No. 32, pp. 6820-33 and Volume 55, No. 11, pp. 25240-81.
Fujiwara, S. And Noguchi, T. (1995). Degradation of Purines: Only Ureidoglycollate Lyase Out of Four
Allantoin-degrading Enzymes is Present in Mammals. The Biochemical Journal 312 (Part 1):315-18.
Thornfeldt, C. (2005). Cosmeceuticals Containing Herbs: Fact, Fiction or Future. Dermatologic Surgery 31
(7 part 2): 873-80.
Young, E.G., Wentworth, H.P. and Hawkins, W.W. (1944). The Absorption and Excretion of Allantoin in Mammals.
J. Pharmacol. Experi. Therapeutics 81: 1-9.

CHROMABOND is a registered trademark of Macherey-Nagel GmbH

ASPEC is a trademark of Gilson, Inc.
NUCLEODUR is a registered trademark of Macherey-Nagel GmbH

Application Note PHA0110, December, 2010

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